Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9

Meizhu Bai^{a, b, c, #},
Dan Liang^{b, c, #},
Yinghua Wang^{b, c},
Qing Li^{b, c},
Yuxuan Wu^{b, c
, ,},
Jinsong Li^{a, b, c
, ,}

a.
School of Life Science and Technology, Shanghai Tech University, Shanghai 200031, China
b.
Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
c.
Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

More Information

Corresponding author: E-mail address: yuxuan.wu@sibcb.ac.cn (Yuxuan Wu); E-mail address: jsli@sibcb.ac.cn (Jinsong Li)

These authors contributed equally to this work.

摘要

- /
- /
- /
Abstract: Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern.
- CRISPR-Cas9 /
- Tissue-specific knockout /
- Spermatogenesis /
- Mouse
These authors contributed equally to this work.
注释:

HTML全文

参考文献(38)

[1]	Ablain, J., Durand, E.M., Yang, S. et al. A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish Dev. Cell, 32 (2015),pp. 756-764
[2]	Baker, M.D., Ezzati, M., Aloisio, G.M. et al. The small GTPase Rheb is required for spermatogenesis but not oogenesis Reproduction, 147 (2014),pp. 615-625
[3]	Bao, J., Ma, H.Y., Schuster, A. et al. Incomplete cre-mediated excision leads to phenotypic differences between Stra8-iCre; Mov10l1(lox/lox) and Stra8-iCre; Mov10l1(lox/Delta) mice Genesis, 51 (2013),pp. 481-490
[4]	Chang, N., Sun, C., Gao, L. et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos Cell Res., 23 (2013),pp. 465-472
[5]	Cong, L., Ran, F.A., Cox, D. et al. Multiplex genome engineering using CRISPR/Cas systems Science, 339 (2013),pp. 819-823
[6]	Dix, D.J., Allen, J.W., Collins, B.W. et al. Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility Proc. Natl. Acad. Sci. USA, 93 (1996),pp. 3264-3268
[7]	Dores, C., Alpaugh, W., Dobrinski, I. Cell Tissue Res., 349 (2012),pp. 691-702
[8]	Doudna, J.A., Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9 Science, 346 (2014),p. 1258096
[9]	Dow, L.E., Fisher, J., O'Rourke, K.P. et al. Nat. Biotechnol., 33 (2015),pp. 390-394
[10]	Eddy, E.M. Role of heat shock protein HSP70-2 in spermatogenesis Rev. Reprod., 4 (1999),pp. 23-30
[11]	Friedland, A.E., Tzur, Y.B., Esvelt, K.M. et al. Nat. Methods, 10 (2013),pp. 741-743
[12]	Gallardo, T., Shirley, L., John, G.B. et al. Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre Genesis, 45 (2007),pp. 413-417
[13]	Gossen, M., Bujard, H. Studying gene function in eukaryotes by conditional gene inactivation Annu. Rev. Genet., 36 (2002),pp. 153-173
[14]	Hsu, P.D., Lander, E.S., Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering Cell, 157 (2014),pp. 1262-1278
[15]	Huszar, J.M., Jia, Y., Reddy, J.K. et al. Med1 regulates meiotic progression during spermatogenesis in mice Reproduction, 149 (2015),pp. 597-604
[16]	Inselman, A.L., Nakamura, N., Brown, P.R. et al. Heat shock protein 2 promoter drives Cre expression in spermatocytes of transgenic mice Genesis, 48 (2010),pp. 114-120
[17]	Jinek, M., Chylinski, K., Fonfara, I. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity Science, 337 (2012),pp. 816-821
[18]	Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K. et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells Bio. Reprod., 69 (2003),pp. 612-616
[19]	Keber, R., Acimovic, J., Majdic, G. et al. Male germ cell-specific knockout of cholesterogenic cytochrome P450 lanosterol 14alpha-demethylase (Cyp51) J. Lipid Res., 54 (2013),pp. 1653-1661
[20]	Lakso, M., Sauer, B., , Lee, E.J. et al. Targeted oncogene activation by site-specific recombination in transgenic mice Proc. Natl. Acad. Sci. USA, 89 (1992),pp. 6232-6236
[21]	Lei, Z., Lin, J., Li, X. et al. Postnatal male germ-cell expression of cre recombinase in Tex101-iCre transgenic mice Genesis, 48 (2010),pp. 717-722
[22]	Li, D., Qiu, Z., Shao, Y. et al. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system Nat. Biotechnol., 31 (2013),pp. 681-683
[23]	Li, S., Lan, Z.J., Li, X. et al. J. Reprod. Infertil., 15 (2014),pp. 122-133
[24]	Li, W., Teng, F., Li, T. et al. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems Nat. Biotechnol., 31 (2013),pp. 684-686
[25]	Mali, P., Yang, L., Esvelt, K.M. et al. Science, 339 (2013),pp. 823-826
[26]	Niu, Y., Shen, B., Cui, Y. et al. Cell, 156 (2014),pp. 836-843
[27]	Ryding, A.D., Sharp, M.G., Mullins, J.J. Conditional transgenic technologies J. Endocrinol., 171 (2001),pp. 1-14
[28]	Sadate-Ngatchou, P.I., Payne, C.J., Dearth, A.T. et al. Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice Genesis, 46 (2008),pp. 738-742
[29]	Shen, Z., Zhang, X., Chai, Y. et al. Dev. Cell, 30 (2014),pp. 625-636
[30]	Stolfi, A., Gandhi, S., Salek, F. et al. Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9 Development, 141 (2014),pp. 4115-4120
[31]	Teves, M.E., Jha, K.N., Song, J. et al. Andrology, 1 (2013),pp. 37-46
[32]	Toyooka, Y., Tsunekawa, N., Takahashi, Y. et al. Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development Mech. Dev., 93 (2000),pp. 139-149
[33]	Wang, H., Yang, H., Shivalila, C.S. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering Cell, 153 (2013),pp. 910-918
[34]	Wu, Y., Liang, D., Wang, Y. et al. Cell Stem Cell, 13 (2013),pp. 659-662
[35]	Wu, Y., Zhou, H., Fan, X. et al. Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells Cell Res., 25 (2015),pp. 67-79
[36]	Yang, H., Wang, H., Shivalila, C.S. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering Cell, 154 (2013),pp. 1370-1379
[37]	Yuan, L., Liu, J.G., Zhao, J. et al. Mol. Cell, 5 (2000),pp. 73-83
[38]	Zhang, M., Zhou, H., Zheng, C. et al. Sci. Rep., 4 (2014),p. 5936